1. Field of the Invention
The present invention relates to methods and compositions for inhibiting CD14 mediated cell activation. More particularly, the present invention relates to molecules that bind the CD14 monocyte antigen at a site which inhibits CD14 mediated cell activation.
2. Description of the Related Art
The correct functioning of a cell depends partly on its ability to communicate with its environment; external stimuli often interact with membrane receptors which, in turn, induce second messengers that ultimately affect transcription factors. The latter then activate or repress the expression of certain genes resulting in a specific pattern of proteins in a given cell. The transcription factor NF-xcexaB (nuclear factor-xcexaB) is induced by a variety of stimuli to contact its DNA-binding motif and regulate a set of genes encoding immunoreceptors, cytokines, and viral proteins. Included among the various factors which can activate NF-xcexaB is lipopolysaccharide (LPS). LPS, in turn, is intimately involved in the induction of the sepsis syndrome, including septic shock, systemic inflammatory response syndrome, and multiorgan failure.
Sepsis is a morbid condition induced by a toxin, the introduction or accumulation of which is most commonly caused by infection or trauma. The initial symptoms of sepsis typically include chills, profuse sweat, irregularly remittent fever, prostration and the like, followed by persistent fever, hypotension leading to shock, neutropenia, leukopenia, disseminated intravascular coagulation, acute respiratory distress syndrome and multiple organ failure.
Lipopolysaccharide, or endotoxin, is a toxic component found in the outer membrane of all gram-negative microorganisms (e.g., Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa). It has been determined that LPS is a potent and pleiotropic stimulus for immune cells, both in vitro and in vivo (Morrison, D. C. and J. L. Ryan, Annu. Rev. Med., 38:417, 1987, Bone, R. C., Ann. Intern. Med., 115:457, 1991). Compelling evidence supports the toxic role of LPS in that all of the pathophysiological effects noted in humans during gram-negative sepsis can be completely duplicated with purified LPS. The mechanism by which this toxic component activates responsive cells is complex and not fully understood. The host response to gram-negative bacterial infection is dependent upon effector cell recognition of these bacteria and/or LPS and involves serum proteins and cell membrane receptors. While the clearance of bacteria and LPS is via endocytosis and phagocytosis by reticuloendothial cells, concomitant activation of the host immune response by LPS results in secretion of cytokines by activated macrophages which can trigger the exaggerated host responses that occur during gram-negative bacterial infection.
The discovery by Tobias, et al. (J. Exp. Med., 164:777, 1986) of a serum protein, identified as LPS binding protein (LBP), that exhibits high affinity binding to LPS (Kd≈10xe2x88x929 Mxe2x88x921), helped to define the fate of LPS once released in vivo. It was demonstrated that this novel protein, with a molecular weight of 60 kD, which is synthesized in the liver is an acute phase serum protein reaching levels of 200 xcexcg/ml in humans. The formation of high affinity LPS/LBP complexes is followed by recognition by macrophages with subsequent release of TNF-xcex1 and other macrophage secretory products (Schumann, R. R., et al., Science, 249:1429, 1990). Additional studies on the effects of LPS complexed with LPB led to the discovery of its specific receptor on the surface of monocytes and macrophages; CD14 (Wright, S. D., et al., Science, 249:1431, 1990). Further analysis with mAbs specific for CD14 revealed that the domain to which one anti-CD14 mAb (3C10; VanVoorhis, W. C., et al., J. Exp. Med., 158:126, 1983) bound was part of, or in close proximity to, the LPS/LBP binding site on CD14. Monoclonal antibody 3C10, by nature of its ability to block LPS/LBP binding to CD14, was capable of inhibiting TNF-xcex1 release in a human whole blood assay, after stimulation with LPS. It is suggested by this discovery that the blocking of a single protein determinant (the ligand binding site on CD14) is sufficient, even in the presence of all other cells, proteins and factors contained in human whole blood, to inhibit TNF-xcex1 release (known to be a key mediator in septic shock) and other macrophage secretory products in response to LPS.
In spite of the advances which have been made in understanding the nature of CD14 mediated cell activation disorders, such as sepsis, considerable need remains for compositions which can be used to inhibit such activation and to diagnose these disorders. The present invention provides such compositions.
This invention provides hybridoma cell lines producing monoclonal antibodies, the monoclonal antibodies being capable of inhibiting CD14 mediated cell activation. Monoclonal antibodies produced by these cell lines also are provided. These monoclonal antibodies are broadly useful in inhibiting NF-xcexaB activation by a ligand which binds to CD14 and would otherwise be capable of inducing NF-xcexaB activation. Biologically active fragments of the monoclonal antibodies are provided. The antibodies and fragments are useful for the detection of the presence of cell surface-associated and soluble CD14 in a sample. Chimeric and CDR grafted antibodies generated from the above monoclonal antibodies are further provided.
Pharmaceutical compositions containing the above biological compositions are provided. These are useful to treat and prevent LPS-associated disorders, such as sepsis.